Hepatitis B Virus (HBV) infection is a worldwide biomedical problem and an improved understanding of the assembly and structure of the virus may help develop new anti-viral therapies. HBV consists of a glycoprotein envelope and an inner nucleocapsid protein called the core antigen (HBcAg). The 183 residues HBcAg was previously expressed in E.coli were it assembles in the bacterial cytoplasm into icosahedral capsids, which contain bound host nucleic acid. Deletion of the polybasic C-terminal 34 residues produces assembly competent protein. The C-terminal truncated protein (Cpe: residues 1-149) does not contain nucleic acid, can be highly purified and is more suitable for structural studies than the full length protein. The structure of Cpe at 1.7 nm resolution, established last year by cryo-electron microscopy and image analysis, was improved to 0.9 nm. This allowed various structural domains to be discerned, especially a four alpha-helical bundle which formed the surface projections of the capsid. To localize other regions of functional and structural importance, we have introduced by site-directed mutagenesis reactive cysteine residues at various sites in the protein. In one example a unique C-terminal cysteine was introduced and specifically labeled with an electron dense gold cluster using sulfhydryl-based chemistry. Following visualized by cryo-electron microscopy, the gold label was located at the inner surface of the capsid under the five-fold and quasi-six-fold vertices of the icosahedral structure. Analysis of full length capsids (residues 1-183) containing bound nucleic acid (RNA) revealed that the RNA contacted the protein shell around the same vertices. These findings highlight the structural and functional importance of the C-terminal region. Recently, Cpe capsids have been crystallized. The crystals appear suitable for X-ray structure determination and this work is underway.